Influenza virus remains an important viral pathogen of significant medical importance. Recently, influenza virus has been classified as a Select Agent Category C because of the ease of the spread of the virus and its ability to cause great morbidity and mortality. Each year in the USA, in the absence of the introduction of pandemic influenza, the virus kills 36,000 people, hospitalizes 114,000 and causes 70 million missed work days and 38 million lost school days. It is estimated the loss to the economy is $3-15 billion. In years of introduction of pandemic influenza virus (1957 and 1967) approximately 70% of the US population were infected and in 1918/19 the estimate of death associated with Spanish influenza range from 20-40 million (1 in 100 people). The outbreaks of highly pathogenic H5N1 avian influenza in 1997 and 2004/6 with the death of millions of chickens and the very high mortality rate of the limited number of infected humans, gives rise to great concern in the event that the avian virus mutates such that it transmits readily from human to human. Current production methods for vaccines means there is a 5-6 month delay between the time of isolation of a new pathogenic strain of human influenza virus and the widespread distribution of the vaccine. Thus, to protect the world population should H5N1 or any other pandemic strain of influenza virus arise, there is an immediate need to understand the entire process of replication of influenza virus in order to discover new targets for the development of anti-viral drugs. In this grant period we will study the mechanism of assembly and budding of influenza virus, a largely unexplored area. We will analyze the requirements for production of virus-like particles (VLPs) with emphasis on the roles of the cytoplasmic tails of the hemagglutinin (HA), neuraminidase (NA) and the M2 ion channel as the available evidence indicates there are differences in requirements among HA subtypes, including H5N1 high pathogenicity avian influenza virus. We will examine the plasma membrane distribution of HA, NA, M2 and mutants of these proteins by immunogold staining and electron microscopy and examine the localization of the viral membrane (M1) protein by using cell-rip procedures and immunogold staining. We will study the assembly of mutant viruses that contain truncations to the M2 cytoplasmic tails to determine the role of M2 in virus assembly. We will seek cellular proteins that interact with the M1 protein that are involved in influenza virus assembly and budding using multiple approaches including generating stable cell lines that express affinity tagged M1 protein, genome-wide shRNA screens and genome-wide yeast two hybrid experiments. M1 protein interacting partners will be confirmed by biochemistry and functionally by siRNA knock-down experiments.